Process for the manufacture of arginine base



Patented Dec. 12, 1950 UNITED STATE PROCESS FOR THE MANUFACTURE OF QARGININE :BASE

Dominic-I. Bernardi, New York, and Ferdinand A. Dostal, Maspeth, N. Y.,assignors to Interchemical Corporation, New York, N. Y., a corporationof Ohio No Drawing.

Application September 22, 1948,

Serial No. 50,654

I the co-pending' application serial No. 624,081, filed October 23,1945, which matured into Patent No. 2,457,117 of December'ZB, 1948, anewmethod is disclosed and claimed for the isolation and preparation of thebasic amino acids lysine, arginine, and histidine from a proteinhydrolysate. There, the three amino'acids are recovered as themonohydrochlorides.

' The subject of the present invention is an improvement in theisolation and recovery of arginine which, by following the hereindisclosed method is obtained as the free base.

According to the aforementioned patent application protein matter ishydrolyzed by means of mineral acids or enzymes. or by means ofcombinations thereof. The resulting hydrolysate containing an amino acidmixture which correspondsin its proportions closely to the amino acidcomposition of the protein'thatwas used in the hydrolysis, after havingbeen neutralized if. such neutralization is needed, and after havingbeen freed from humic bodies, unhydrolyzed protein matter and othersolids or salts as may be present, and from ammonia, is subjected to anion-exchange process. For this purpose columns of natural or syntheticzeolites are used which are characterized by permitting ion-exchange inthe ,so-called salt cycle, 1. e. base exchangers which substitute onecationagainst another.

By means of adjusting the pH of the hydrolysate to about 8 and passingit through a first ion-exchange column of the described type, the twobasic amino acids lys ne and arginine are quantitatively adsorbed in thecolumn. In this manner it is possible to separate them from hi'stidine,the third member of the group of basic amino'acids and from all otheramino acids of the hydrolysate. After acidifying the effluent from thefirst column to pH 6 or less, the efliuent is passed through a secondion-exchange column of the same type in order to separate the histidinefrom the other amino acids.

The lysine and arginine are eluted by means of, say, a sodium chloridesolution. The eluate from the column, containing lysineand argininemonohydrochloride, is freed of most of the sodium chloride byalternately concentrating in vacuo and removing crystall zed sodiumchoride from the hot solution. Having concentrated the efiluent in thismanner, until it contains about 150 gr. lysine hydrochloride and aproportional amount of arginine monohydrochloride per liter, an equalvolume of methyl alcohol is added and salt, which again crystal izes, isremoved after heating the extract- Refrigeration overnight, preferablywith mechanical stirring, causes lysine monohydrochloride tocrystallize. After separating the lysine-monohydrochloride the remainingalcoholic mother liquor contains, aside from the arginine, salts,varying amounts of residual 2 Claims. (01. 260-529) lysine, anddifierent impurities. H Upon" partial evaporation or evaporation todryness the mother liquor yields crude, technical gradeargininemonohydrochloride. A method of recovering this I amino acid inpure formiconsists in precipitating (a) Rendering the mother-liquoralkaline to a specific pH,

(b) Employing a specific base for the pH adjust-.

ment.

Of the various bases, such assodium hydroxide, ammonium hydroxide,potassium hydroxide, calcium hydroxide and sodium carbonate, only sodiumhydroxide is satisfactory, The pH to which the mother liquor must beadjusted may vary from batch to batch but, as -a rule, falls between10.5 and 13. If the pH is appreciably lower or higher the'yield isusually nil. The following table serves to illustrate the relationbetween the pH value to which the mother liquor has been adjusted andthe per cent arginine recovered.

' Per Cent PH recovery l2.v 50 33. 0 12. 21. 4 13. O0 0. 0

Although the free arginine base can be efliciently recovered from theaqueous solution, we find that the addition of one to two volumes methylor ethyl alcohol improves the yield and the purity of the recoveredarginine base.

The following example will illustrate the steps of the procedure.

266 pounds of hogs' blood from an'abattoir; containing 18 percent or 48pounds of protein matter, are hydrolized under refluxing with 1126poundsof 98 percent sulfuric acid, which has been diluted beforehand, ifnecessary, so that the initial sulfuric acid concentration is about 24per cent by volume or 35 per cent by weight. After IG-hours ofrefluxing. the batch is diluted with an equal volume of water andbrought to a pH of about 9.0-by means of adding 36 gallons of limeslurry. containing approximately 100 pounds of slaked lime. Calciumsulfate which forms upon the addition of the lime is removed by means ofa filter press. A thorough washing of the filter cake, in order torecover as much as possible of the extract, is achieved by following thefiltrate with so much hot water that a total of 172 gallons of processsolution results, of an extract concentration of about 3 per cent. Atthis point the process solution is preferably reduced to about 60gallons of 8 to 9 per cent extract by means of concentrating in vacuowhereby, incidentally, any ammonia is removed that may have formedduring the hydrolysis.

[Small amounts of calicum cipitate during the vacuum concentration canbe removed by either filtration or by means of a centrifugal. insolution, are eliminated after the careful addition of molar equivalentsof oxalic acid and barium hydroxide. The extract is then subjected to anion-exchange process, preferably carried outwith the aid of anion-exchange column filled with a natural or synthetic ion-exchangematerialwhich is distinguished by reacting in the so-called salt cycle.Ion-exchange material, sold under the trade name Decalso is a suitableproduct for these specific purposes.

After having adjusted the extract to alkalinity of a pH from 7.5 to 8.0,the extract is pumped, in this specific instant at a rate of about 48gallons per hour, through a column containing a sufficiently largeamount of the prepared ionexchange material. 120 pounds of theaforementioned Decalso allow for an adequate safety margin m. theadsorption of substantially all the lysine and arginine contained in thehydrolysate.

,Hav-ing finished the adsorption cycle, the column is rinsed until theoutflow is free of amino acids. Following this rinse, lysine andarginine areeluted by means of a per cent sodium chloride brine of which70 gallons are pumped through .the column at a rate of approximately 1.2gallons per minute. The eluate, containing lysineand argininmonohydrochloride, is freed of the larger part of the sodium chloride byalternately concentrating in vacuo and removing crystallized sodiumchloride from the hot solution. Having reduced, in this manner, thevolume of the concentrate to two gallons or less, an equal volume oi.methyl alcohol is added and salt, which crystallizes upon this addition,is removed, once more, after having heated the extract. Refrigerationovernight, preferably with mechanical stirring, causes lysinemonohydrochloride tocrystallize which is separated from the solution.The solution is freed from the methanol by means of a vacuumdistillation which permits the recovery of the alcohol. The resulting7000 cc. of arginine mother liquor are made alkaline to a pH of 11.63 byadding 6900 cc. of 6 N sodium hydroxide.

' The pH value of 11.63 was determined empirically as the optimum forthe recovery of arginine from this particular batch. For this pur posesmall aliquots were adjusted to various pH readings within the statedrange of pH 10.5 to 13 and the arginine yield of each sample determinedin the described manner. Upon the addition of the sodium hydroxide agelatinous precipitate forms, containing iron hydroxide and magnesiumhydroxide which stems in part from the hemo- Calcium and sulfate ions,remaining globin of the blood and from the lime' used in neutralization.This precipitate is filtered off and washed with about 3500 cc. of hotwater.

The filtrate is then concentrated in vacuo to a volume of about 270000., heated in a boiling water bath for about minutes and. filtered hotthrough a steam heated Biichner funnel to remove crystallized salt. Thesalt is washed with about 450 cc. of water which is added to thefiltrate. The latter is then refrigerated overnight with stirring. Wefind it advantageous to seed the solution with a crystal of argininebase. The next morning the precipitate which has formed is filtered offand washed thoroughly with methanol, sucked dry and dried in a vacuumoven at 70. C. The yield is slightly more than 40% of the, argininepresent in the mother liquor. After recrystallization the arginine baseshows: less than 0.1% of lysine, 0.0% ash, 31.85% N. One modification ofthe procedure which results in somewhat higher yields of a still purerproduct is to add 1 to 3 volumes'of methyl alco-f hol to the concentrateafter the removal of the salt. After the addition of 2 volumes ofmethanol to a similar batch the yield exceeds GO% of the theoretical andthe product shows, after rerefrigerating,

crystallization, an optical rotation of (a) '=25.O in 1 N HCl,indicating pure 1 arginine.

If particular emphasis is placed upon the preparation of ch mically purearginine rather than other amino acids, a protein should be selectedwhich is relatively high in arginine.

Gelatine, for instance, is about 25 per cent highe in argininethan bloodprotein. Having illustrated our new method, we claim: 1. The recovery offree arginine base from a mother liquor containing arginine, residuallysine,

and impurities, by adjusting the mother liquor,

2'. The recovery of free arginine base from a mother liquor containingarginine, residual lysine, and impurities, by adjusting the mother.liquor to alkalinity of a pH above 10.5 and below 13 with.- sodiumhydroxide, removing any precipitatef which forms upon the additionofthesodium hydroxide, removing crystallized salt from the hot.

solution after concentration in vacuo to 15-25 gr. arginine per cc.,seeding, refrigerating the solution with stirring, and separating thecrystallized arginine base.

DOMINIC J. BERNARDI. FERDINAND A. DOSTAL.

REFERENCES CITED The following references are of record in the file ofthis patent: v

UNITED STATES PATENTS Number Name Date 1,973,574 Marshall Sept. 11, 19342,347,220 S'hildneck Apr. 25, 1944 2,373,342 Royal Apr. 10, 19452,433,219 Hoglan Dec. 23, 1947 OTHER REFERENCES.

Block: Archives of Biochem., vol. 11, pp. 236-

1. THE RECOVERY OF FREE ARGININE BASE FROM A MOTHER LIQUOR CONTAININGARGININE, RESIDUAL LYSINE, AND IMPRUITIES, BY ADJUSTING THE MOTHERLIQUOR TO ALKALINITY OF A PH ABOVE 10.5 AND BELOW 13 WITH SODIUMHYDROXIDE, REMOVING ANY PRECIPITATE WHICH FORMS UPON THE ADDITION OF THESODIUM HYDROXIDE, REMOVING CRYSTALLIZED SALT FROM THE HOT SOLUTION AFTERCONCENTRATION IN VACUO, REFRIGERATING, AND SEPARATING THE CRYSTALLIZEDARGININE BASE.